Va14/ NKT cells are a unique T cell subset that recognizes glycolipids presented by CD1d molecules. Recent evidence suggests that a major physiologic function of these cells is the response to microbes or microbial products. This Va14/ NKT cell-mediated immune reaction can occur in two ways. First, the relatively invariant T cell antigen receptor (TCR) these cells express can directly recognize some bacterial glycolipids. Second, Va14/ NKT cells can even respond to those bacteria that do not have antigens for the invariant TCR;we call this indirect recognition or sensing of these microbes. In some cases, this activation can occur through the stimulation of antigen presenting cells (ARC) by TLR ligands, leading to IL-12 and IL- 18 secretion, which activates Va14/ NKT cells even in the absence of self-antigen. The experiments in this proposal are designed to achieve a better molecular and cellular understanding of the indirect recognition process. We will confirm that ligands for a limited set of TLRs, namely those for TLR4 and TLR9, are capable of causing Va14/ NKT cell IFNy release. We will identify the ARC responsible for activating Va14/ NKT cells after exposure to E. coll LPS or CpG ODN, and will assess their expression of the relevant TLRs and their ability to produce the required activating cytokines. The role of IFNy receptor signaling in enhancing the IL-12/IL-18 mediated response will be determined, as will the regulation of IL-12R and IL-18R expression by Va14/'NKT cells. The effects of these agents on the expression of CD1d by the relevant ARC will also be determined. In some other cases, microbial products may induce the increased expression of self-antigens recognized by the invariant TCR, and or increased expression of CD1d. Furthermore, it is possible that APC-derived cytokines can synergize with TCR-mediated signals delivered by self-antigens presented by CD1d. In the response to Salmonella LPS and to Schistosome egg antigen preparations, self- antigen presentation has been implicated in activating Va14/ NKT cells. We will carry out experiments to determine if either of these agents affects CD1d expression, and to more rigorously test for self-antigen presentation. These experiments will yield important insights into the normal role of Va14/ NKT cells in combating different types of infections, and therefore the findings could lead to novel vaccination strategies based on the innate-like properties of /NKT cells.